1. Field
The present disclosure relates to methods and compositions for nucleic acid amplification, particularly in the presence of nucleic acid amplification inhibitors.
2. Description of the Related Art
The amplification of nucleic acid from a biological material is affected by cellular lysis and the presence of various nucleic acid amplification inhibitory substances. Therefore, removing nucleic acid amplification inhibitors from biological materials improves nucleic acid amplification and assays related thereto, including molecular diagnostics.
Biological materials include, for example, mucus, blood, stool, and tissue samples from a subject. Nucleic acid amplification from a stool sample is particularly challenging, since sample variation is large and because nucleic acid amplification inhibitors, particularly heme, bilirubin, bile salt, and a variety of complex polysaccharides, may exist in the sample.
Nucleic acid amplification inhibitors can be removed from a biological material during pre-treatment or post-treatment of the sample. Removal of nucleic acid amplification inhibitors during pre-treatment of the sample (i.e., before preparation of the sample for nucleic acid amplification) is complicated, can be affected heavily by reactions, binding, and elution buffers used in the pre-treatment process, and may cause loss of target cells. Removal of nucleic acid amplification inhibitors during post-treatment of the sample (i.e., after preparation of the sample for nucleic acid amplification) may involve, for example, the addition of a nucleic acid amplification inhibitor removal substance to a polymerase chain reaction (PCR) master mix. No critical loss of target cells or DNA is observed in post-treatment. However, post-treatment is largely ineffective at removing nucleic acid amplification inhibitors. The use of various chemical substances (acetamide, betaine, dextran, DMSO, formamide, glycerol, PEG, PVP-10 etc.), detergents (tween, SDS etc.), and biomolecules (gp32 single strand binding protein, proteinase inhibitor etc.) have been studied but their effects are largely insufficient. Only BSA has been shown to have a slight effect on the removal of nucleic acid amplification inhibitors in some samples.
Therefore, improved method of amplifying nucleic acids from biological materials is needed.